Trichoderma reesei glucoamylase and homologs thereof

ABSTRACT

The present invention is related to glucoamylases having at least 80% sequence identity to a Trichoderma glucoamylase having the sequence of SEQ ID NO: 4 and biologically functional fragments thereof. The invention is also related to DNA sequences coding for the glucoamylases, vectors and host cells incorporating the DNA sequences, enzyme compositions and methods of using the glucoamylases in various applications.

RELATED APPLICATIONS

The present application is a continuation in part application ofapplication Ser. No. 11/136,244 filed May 24, 2005, which claimspriority to provisional patent application Ser. No. 60/647,925 filedJan. 28, 2005, International Patent Application PCT/US04/041276 filedDec. 9, 2004; International Application PCT/US04/040040, filed Nov. 30,2004; provisional application Ser. No. 60/605,437, filed Aug. 30, 2004,now abandoned and provisional application Ser. No. 60/575,175, filed May27, 2004, now abandoned, and claims priority to International PatentApplication PCT/US05/0018212 filed May 24, 2005; which claims priorityto provisional applications Ser. No. 60/647,925 filed Jan. 28, 2005,Ser. No. 60/605,437 filed Aug. 30, 2004, now abandoned and Ser. No.60/575,175 filed May 27, 2004, now abandoned and InternationalApplication PCT/US04/040040, filed Nov. 30, 2004 the contents of whichare fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to new glucoamylases useful for theproduction of glucose and other end products from starch. Theglucoamylases are suitable for use in various processes and areparticularly suitable for use under conditions of conventional hightemperature starch processing and under conditions of non-cook or lowtemperature starch processing.

BACKGROUND OF THE INVENTION

Glucoamylase enzymes (α-1,4-glucan glucohydrolases, E.C.3.2.1.3.) arestarch hydrolyzing exo-acting carbohydrases. Glucoamylases catalyze theremoval of successive glucose units from the non-reducing ends of starchor related oligo and polysaccharide molecules and can hydrolyze bothlinear and branched glucosidic linkages of starch (amylose andamylopectin).

Glucoamylases are produced by numerous strains of bacteria, fungi, yeastand plants. Particularly interesting glucoamylases are fungal enzymesthat are extracellularly produced, for example from strains ofAspergillus (Boel et al., (1984) EMBO J. 3:1097-1102; Hayashida et al(1989) Agric. Biol. Chem. 53:923-929; U.S. Pat. No. 5,024,941; U.S. Pat.No. 4,794,175; and WO 88/09795), Talaromyces (U.S. Pat. No. 4,247,637;U.S. Pat. No. 6,255,084 and U.S. Pat. No. 6,620,924), Rhizopus (Ashikariet al. (1986) Agric. Biol. Chem. 50:957-964; Ashikari et al. (1989) App.Microbiol. and Biotech. 32:129-133 and U.S. Pat. No. 4,863,864),Humicola (WO05/052148 and U.S. Pat. No. 4,618,579) and Mucor(Houghton-Larsen et al., (2003) Appl. Microbiol. Biotechnol., 62:210-217). Many of the genes, which code for these enzymes have beencloned and expressed in yeast and fungal cells.

Commercially glucoamylases are very important enzymes that have beenused in a wide variety of applications requiring the hydrolysis ofstarch. Glucoamylases are used for the hydrolysis of starch to producehigh fructose corn sweeteners, and corn sweeteners comprise over 50% ofthe US sweetener market. In general, starch hydrolyzing processesinvolve the use of alpha amylases to hydrolyze the starch to dextrinsand glucoamylases to hydrolyze the dextrins to glucose. The glucose isthen converted to fructose by other enzymes such as glucose isomerases.Glucose produced by glucoamylases can also be crystallized or used infermentations to produce other end-products, such as citric acid,ascorbic acid, glutamic acid, 1,3 propanediol and others. Glucoamylasesare used in alcohol production, such as beer production and sakeproduction. Glucoamylases also find use in the production of ethanol forfuel and for consumption. Recently, glucoamylases have been used inlow-temperature processes for the hydrolysis of granular (non-cooked)starch. Glucoamylases are also used in the preparation of animal feedsas feed additives or as liquid feed components for livestock animals.

Although glucoamylases have been used successfully for many years, aneed still exists for new useful glucoamylases. The present invention isbased upon the finding of novel glucoamylases suitable for use invarious applications and particularly starch conversion processes.

SUMMARY OF THE INVENTION

The invention is directed to an isolated DNA sequence encoding aglucoamylase having at least 80% identity to SEQ ID NO: 4.

In another embodiment, the invention is directed to an enzyme havingglucoamylase activity comprising the amino acid sequence of SEQ ID NO: 4or substantially homologous sequences thereto and allelic variants andbiologically functional fragments thereof.

In another embodiment, the invention is related to an isolated DNAsequence encoding a Trichoderma reesei glucoamylase including the nativegene sequence and biologically functional fragments thereof.

In another embodiment, the invention is direct to vectors comprising aDNA sequence encoding the glucoamylases encompassed by the invention.

In another embodiment, the invention is directed to stable transformedfungal host cells, particularly Trichoderma and Aspergillus host cellsand methods for the expression of the glucoamylase therefrom.

In another embodiment, the invention is directed to a culture mediumincluding a glucoamylase encompassed by the invention and enzymepreparations obtained from the growth or culture of transformed hostsand the use of the enzyme preparations.

In another embodiment, the invention is directed to starch conversionprocesses using the enzyme preparations of the invention. In someembodiments, the glucoamylase will be used in a process of convertingstarch or partially hydrolyzed starch into a syrup containing dextrose.In other embodiments, the glucoamylase will be used in a process forproducing specialty syrups. In further embodiments, the glucoamylasewill be used in a fermentation to produce end products, such as alcoholsand particularly ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B shows the genomic DNA sequence (SEQ ID NO: 1) coding for theTrichoderma reesei glucoamylase of FIG. 3.

FIGS. 2A-B shows the intronless DNA sequence (SEQ ID NO: 2) coding forthe Trichoderma reesei glucoamylase of FIG. 3.

FIG. 3A shows the deduced amino acid sequence (SEQ ID NO: 3) of theTrichoderma reesei glucoamylase having 632 amino acids, wherein

the signal sequence (SEQ ID NO: 38) is in bold and is represented byresidue positions 1-20;

the prosequence (SEQ ID NO: 39) is in bold and underlined andrepresented by residue positions 21-33;

the catalytic domain (SEQ ID NO: 40) is represented by residue positions34-486;

the linker region (SEQ ID NO: 41) is in italics and represented byresidue positions 487-523; and In other embodiments, the starch bindingdomain is a fragment of the starch binding domain of SEQ ID NO: 4.Preferably a fragment will encompass at least 90, at least 80 or atleast 70 amino acid residues of the starch binding domain of SEQ ID NO:4.

the starch binding domain (SEQ ID NO: 42) is in italics and underlinedand represented by residue positions 524-632.

The N-terminal amino acid residue of the mature protein represented byresidue position 34 is serine.

FIG. 3B shows the deduced mature protein sequence (SEQ ID NO: 4) of theTrichoderma reesei glucoamylase of FIG. 3A. The mature protein sequenceincludes the catalytic domain, which is underlined (SEQ ID NO: 40), thelinker region (SEQ ID NO: 41) and starch binding domain (SEQ ID NO: 42).

FIG. 4 shows the genomic DNA sequence having 2154 bp (SEQ ID NO: 5)coding for the Hypocrea citrina var. americana glucoamylase (GA102) (SEQID NO: 6).

FIG. 5 shows the genomic DNA sequence having 2152 bp (SEQ ID NO: 7)coding for the Hypocrea vinosa glucoamylase (GA104) (SEQ ID NO: 8).

FIG. 6 shows the genomic DNA sequence having 2158 bp (SEQ ID NO: 9)coding for a Trichoderma sp. glucoamylase (GA105) (SEQ ID NO: 10).

FIG. 7 shows the genomic DNA sequence having 2144 bp (SEQ ID NO: 11)coding for a Hypocrea gelatinosa glucoamylase (GA107) (SEQ ID NO: 12).

FIG. 8 shows the genomic DNA sequence having 2127 bp (SEQ ID NO: 13)coding for a Hypocrea orientalis glucoamylase (GA108) (SEQ ID NO: 14).

FIG. 9 shows the genomic DNA sequence having 2139 bp (SEQ ID NO: 15)coding for a Trichoderma konilangbra glucoamylase (GA109) (SEQ ID NO:16).

FIG. 10 shows the genomic DNA sequence having 2088 bp (SEQ ID NO: 28)coding for Trichoderma sp. glucoamylase (GA113) (SEQ ID NO: 29).

FIG. 11 shows the genomic DNA sequence having 2141 bp (SEQ ID NO: 30)coding for a Trichoderma harzianum glucoamylase (GA103) (SEQ ID NO: 31).

FIG. 12 shows the genomic DNA sequence having 2131 bp (SEQ ID NO: 32)coding for a Trichoderma longibrachiatum glucoamylase (GA124) (SEQ IDNO: 33).

FIG. 13 shows the genomic DNA sequence having 2151 bp (SEQ ID NO: 34)coding for Trichoderma asperellum glucoamylase (GA127) (SEQ ID NO: 35).

FIG. 14 shows the genomic DNA sequence having 2142 bp (SEQ ID NO: 36)coding for Trichoderma strictipilis glucoamylase (GA128) (SEQ ID NO:37).

FIGS. 15A-I shows the putative amino acid sequences for glucoamylasesencoded by the DNA sequences of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 28, 30,32, 34 and 36, which correspond to the amino acid sequences of SEQ IDNOs: 6, 8, 10, 12, 14, 16, 29, 31, 33, 35 and 37 respectively, whereinthe leader peptide is in bold and the prosequence is underlined and inbold for each protein. The mature protein sequence which excludes theleader and prosequence for each protein is also represented as SEQ IDNO: 17 for (1) GA102; SEQ ID NO: 18 for (2) GA104; SEQ ID NO: 19 for (3)GA105; SEQ ID NO: 20 for (4) GA107; SEQ ID NO: 21 for (5) GA108; SEQ IDNO: 22 for (6) GA109; SEQ ID NO: 43 for (7) GA113; SEQ ID NO: 44 for (8)GA103; SEQ ID NO: 45 for (9) GA124; SEQ ID NO: 46 for (10) GA127 and SEQID NO: 47 for (11) GA128.

FIG. 16 illustrates the SDS-PAGE gel used for determining MW of thepurified TrGA, wherein lane 1 exhibits the TrGA and lane 2 exhibits themolecular weight marker SeeBlue Plus 2 (Invitrogen).

FIG. 17A is a plasmid map of T. reesei expression vector, pTrex3g.

FIG. 17B is a plasmid map that includes the T. reesei expression vectorpNSP23, wherein the TrGA gene is cloned into pTrex3g.

FIG. 18 shows (A) the % relative GA activity of the TrGA at 37° C. frompH 3-8 and (B) the % relative GA activity of the TrGA at pH 4.0 from 25°C. to 78° C. and reference is made to example 4.

FIG. 19 illustrates the SDS-PAGE gel used for determining secretion ofsubstantially homologous glucoamylases in the Trichoderma host strain(1A52), wherein the band at about 62 kDa represents glucoamylase andlane 1 represents GA104, lane 2 represents GA105; lane 3 representsGC107; lane 4 represents GA109; lane 5 represents TrGA; lane 6represents a Trichoderma reesei control host strain (1A52); and lane 7represents a standard molecular weight marker.

FIG. 20(A) illustrates the amino acid sequence (SEQ ID NO: 26) for anAspergillus niger glucoamylase which includes the leader sequence. TheN-terminal amino acid residue of the mature protein is represented byresidue position 25, A (alanine); the linker region is underlined andthe starch binding domain is in italics. (B) illustrates the amino acidsequence for an Aspergillus kawachi alpha amylase (SEQ ID NO: 27) whichincludes the leader sequence, wherein the leader sequence is in bold andunderlined and is represented by amino acid residues 1-21; the linkerregion is underlined and the starch binding domain is in italics. Themature protein includes the catalytic domain, the linker and the starchbinding domain.

DETAILED DESCRIPTION OF THE INVENTION

In some aspects, the present invention relies on routine techniques andmethods used in the field of genetic engineering and molecular biology.The following resources include descriptions of general methodologyuseful in accordance with the invention: Sambrook et al. Eds., MOLECULECLONING: A LABORATORY MANUAL (3^(rd) Ed. 2000); Kriegler M. Ed., GENETRANSFER AND EXPRESSION: A LABORATORY MANUAL (1990); and Ausubel et al.Eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (5^(th) Ed. 2002). Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the invention, thepreferred methods and materials are described below.

Unless defined otherwise herein all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton et al., etal., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2^(nd) Ed, JohnWiley and Sons, NY (1994) and Hale and Margham, THE HARPER COLLINSDICTIONARY OF BIOLOGY (1991) Addison Wesley Pub. Co. provides one ofskill with dictionaries of many of the terms used in describing thisinvention.

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications referred to herein are expressly incorporated by reference.

The singular forms “a”, “an” and “the” include the plural referencesunless the content clearly dictates otherwise. Thus for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should be noted that the term “or” isgenerally employed in the sense including “and/or” unless the contentclearly dictates otherwise.

Numeric ranges are inclusive of the numbers of the ranges.

Unless otherwise indicated, nucleic acids are written left to right 5′to 3′ orientation; amino acids sequences are written left to right inamino carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention, which can be had by reference to thespecification as a whole.

Definitions

The term “glucoamylase” refers to the amyloglucosidase class of enzymes(E.C. 3.2.1.3, glucoamylase, 1,4-alpha-D-glucan glucohydrolase). Theseenzymes release glucosyl residues from the non-reducing ends of amyloseand amylopectin molecules.

The phrase “having granular starch hydrolyzing activity” means an enzymethat is capable of hydrolyzing starch in granular form.

The phrase “Trichoderma/Hypocrea family cluster” means a member of theFamily Hypocreaceae including several anamorphs as Trichoderma andGliocladium of the Order Hypocreales, Phylum Ascomycota and reference ismade to Chapter 12, Alexopoulos, C. J., et al., in INTRODUCTORY MYCOLOGY4^(th) Edition, John Wiley & Sons, NY 1996.

The terms “nucleic acid sequence” and “polynucleotide” maybe usedinterchangeably herein. The term encompasses genomic DNA, intronlessDNA, synthetic origins or combinations thereof.

The term “intron” means an intervening DNA sequence that is transcribedbut is removed from within the transcript by splicing together thecoding sequences of the mature protein.

The term “isolated nucleic acid sequence” means a nucleic acid sequence,which is essentially free of other nucleic acid sequences.

The term “biologically functional fragments of a sequence” (e.g.biologically functional fragments of SEQ ID NO: 4) means a polypeptidehaving glucoamylase activity and one or more amino acid residues deletedfrom the amino and/or carboxyl terminus of the amino acid sequence.

The term “vector” means a polynucleotide sequence designed to introducenucleic acids into one or more cell types.

The term “expression vector” means a DNA construct comprising a nucleicacid sequence, which is operably linked to a suitable control sequencecapable of effecting expression of the nucleic acid sequence in asuitable host. Suitable control sequences include promoters to effecttranscription, operator sequences, sequences encoding suitable ribosomebinding sites on the mRNA, enhancers and/or termination sequences.

The term “promoter” means a regulatory sequence involved in binding RNApolymerase to initiate transcription of a gene.

The term “operably linked” refers to juxtaposition wherein theelopements are in an arrangement allowing them to be functionallyrelated. For example, a promoter is operably linked to a coding sequenceif it controls the transcription of the sequence.

The term “an isolated polypeptide” means a polypeptide that isessentially free of other non-glucoamylase polypeptides. An isolatedpolypeptide may be at least 20% pure, at least 40% pure, at least 60%pure, at least 70% pure, at least 80% pure, at least 90% pure, at least95% pure as determined by SDS-PAGE.

The term “signal sequence” means a sequence of amino acids bound to theN-terminal portion of a protein, which facilities the secretion of themature form of a protein outside the cell. The definition of a signalsequence is a functional one. The mature form of the extracellularprotein lacks the signal sequence, which is cleaved off during thesecretion process. The terms “signal sequence”, signal peptide” and“leader peptide” may be used interchangeability herein. In general thesignal sequence refers to the nucleotide sequence and the term leaderpeptide refers to the amino acid sequence.

The terms “protein” and “polypeptide” are used interchangeably herein.The conventional one-letter or three-letter code for amino acidsresidues is used herein.

The term “catalytic domain” refers to a structural region of apolypetide, which contains the active site for substrate hydrolysis.

The term “linker” refers to a short amino acid sequence generally havingbetween 3 and 40 amino acids residues that covalently bind an amino acidsequence comprising a starch binding domain with an amino acid sequencecomprising a catalytic domain.

The term “starch binding domain” refers to an amino acid sequence thatbinds preferentially to a starch substrate.

The term “allelic variants” means any of two or more alterative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation and may result in polymorphism betweenpopulations. An allelic variant of a polypeptide is a polypeptideencoded by an allelic variant of a gene.

The term “host cell” or “host strain” means a suitable host for anexpression vector or DNA construct comprising a polypeptide encoding aglucoamylase encompassed by the invention. Suitable host cells are usedadvantageously in the recombinant production of the glucoamylasesencompassed by the invention.

As used herein the term “derived from” used in connection with apolynucleotide or polypeptide means the polypeptide or polynucleotide isnative to the microorganism.

The term “heterologous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that does not naturally occur in ahost cell.

The term ” endogenous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that occurs naturally in a hostcell.

The term “expression” means the process by which a polypeptide isproduced based on the nucleic acid sequence of a gene.

The term “over expression” means the process of expressing a polypeptideis a host cell wherein a polynucleotide has been introduced the hostcell.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell means transfection, transformation or transductionand includes reference to the incorporation of the nucleic acid sequenceinto a host cell.

The term “granular starch” refers to raw uncooked starch (e.g. granularstarch that has not been subject to gelatinization).

The term “starch” refers to any material comprised of the complexpolysaccharide carbohydrates of plant, comprised of amylose andamylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be any number.

The term “gelatinization” means the solubilization of a starch moleculeby cooking to form a viscous suspension. The phrase “below thetemperature of gelatinization” refers to a temperature less than thetemperature which starts gelatinization.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium. In oneembodiment, culturing refers to fermentative bioconversion of a starchsubstrate to an end-product (typically in a vessel or reactor).Fermentation is the enzymatic and anaerobic breakdown of organicsubstances by microorganisms to produce simpler organic compounds. Whilefermentation occurs under anaerobic conditions it is not intended thatthe term be solely limited to strict anaerobic conditions asfermentation also occurs in the presence of oxygen.

The term “end-product” refers to any carbon source derived moleculeproduct which is enzymatically converted from a starch substrate.

The term” enzymatic conversion” refers to the modification of asubstrate by enzyme action.

The term “specific activity” means an enzyme unit defined as the numberof moles of substrate converted to product by an enzyme preparation perunit time under specific conditions. Specific activity is expressed asunits (U)/mg or protein.

The term “monosaccharide” means a monomeric unit of a polymer such asstarch wherein the degree of polymerization (DP) is 1 (e.g., glucose,mannose, fructose and galactose).

The term “disaccharide” means a compound that comprises two covalentlylinked monosaccharide units (DP2). The term encompasses, but is notlimited to such compounds as sucrose, lactose and maltose.

The term “a DP>3” means polymers with a degree of polymerization greaterthan 3.

The term “oligosaccharide” means a compound having 2-10 monosaccharideunits joined in glycosidic linkages.

The term “polysaccharide” means a compound having multiplemonosaccharide units joined in a linear or branched chain. In someembodiments the term refers to long chains with hundreds or thousands ofmonosaccharide units. Typical examples of polysaccharides are starch,cellulose and glycogen.

As used herein the term “dry solids content (DS or ds)” refers to thetotal solids of a slurry in % on a dry weight basis.

The term “milling” refers to the breakdown of cereal grains to smallerparticles. In some embodiments the term is used interchangeably withgrinding.

The term “dry milling” refers to the milling of dry whole grain, whereinfractions of the grain such as the germ and bran have not been purposelyremoved.

As used herein the terms “distillers dried grain (DDG)” and “distillersdried grain with solubles (DDGS)” refer to useful co-products of grainfermentation processes.

The term “DE” or “dextrose equivalent” is an industry standard formeasuring the concentration of total reducing sugars, calculated asD-glucose on a dry weight basis. Unhydrolyzed granular starch has a DEthat is essentially 0 and D-glucose has a DE of 100.

The term “sugar syrup” refers to an aqueous composition containingsoluble carbohydrates. In one embodiment, the sugar syrup is a syrupcontaining glucose.

Trichoderma Reesei Glucoamylase Amino Acid Sequences

A glucoamylase derived from Trichoderma reesei QM6a (ATCC, Accession No.13631) has been cloned as further described in detail in Example 1.According to the invention the full length glucoamylase derived fromTrichoderma reesei is illustrated in FIG. 3 and has an amino acidsequence of SEQ ID NO: 3. The mature protein sequence of the Trichodermareesei glucoamylase, (SEQ ID NO: 4) is represented by amino acidresidues 34-632 of FIG. 3.

This invention relates to an isolated enzyme having glucoamylaseactivity comprising the sequence shown in SEQ ID NO: 4 or an enzyme withglucoamylase activity being substantially homologous thereto.

In some embodiments, the invention is related to a glucoamylasecomprising the sequence shown in SEQ ID NO: 3 or an enzyme withglucoamylase activity being substantially homologous thereto. Theglucoamylase of SEQ ID NO: 3 includes the signal sequence of theglucoamylase obtained from Trichoderma reesei.

In some embodiments the invention is related to a polypeptide havingglucoamylase activity comprising the catalytic domain of theglucoamylase of SEQ ID NO: 4, which is also represented by SEQ ID NO:40.

In other embodiments, the invention is related to a starch bindingdomain having at least 90%, at least 95%, at least 97%, and at least 98%sequence identity to the starch binding domain of the glucoamylaseillustrated in SEQ ID NO: 4. In some embodiments, the starch bindingdomain encompasses the sequence of residue position 524 to residueposition 632 of SEQ ID NO: 4 and is represented by SEQ ID NO: 42.

In other embodiments, the starch binding domain is a fragment of thestarch binding domain of SEQ ID NO: 4. Preferably a fragment willencompass at least 90, at least 80 or at least 70 amino acid residues ofthe starch binding domain of SEQ ID NO: 4.

Homology of the Protein Sequence

The homology between two glucoamylases may be determined by the degreeof identity between the amino acid sequences of two protein sequences. Apolypeptide or polynucleotide having a certain percent of identity withanother sequence (i.e. 80%, 90%, and 95%) means that when aligned, thatpercent of bases or amino acid residues are the same in comparing thetwo sequences. This alignment and percent homology or identity can bedetermined by using any suitable software program known in the art. Forexample suitable programs are described in CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Ausubel et al., eds 1995, Chapter 19). Preferredprograms include GCG Pileup program (Wisconsin Package, Version 8.1 and10.0), FASTA, BLAST and TFASTA. Another preferred alignment program isALIGN or ALIGN Plus (Dayhoff (1978) in ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE 5: Suppl. 3 (National Biomedical Research Foundation)) FurtherBLASTP, BLASTN and BLASTX algorithms can be used (Altschul et al.,(1990) J. Mol. Biol. 215:403-410). Other useful methods includeClustralW (Thompson et al., (1997) Nucleic Acid Research 25:4876-4882)using software provide by DNASTAR (Madison Wis.). Also reference is madeto Needleman et al., (1970) J. Mol. Biol. 48:443, Smith et al., (1981)Adv. Appl. Math. 2: 482, Smith et al., (1997) Meth. Mol. Biol.70:173-187 and Pearson et al., (1988) Proc. Natl. Acad. Sci. 85:24444.

According to the invention a “substantially homologous” amino acidsequence exhibits glucoamylase activity and at least 80% identity, atleast 83%, at least 85%, at least 87%, at least 90%, at least 93%, atleast 95%, at least 97%, at least 98% and at least 99% identity with thesequence illustrated in SEQ ID NO: 4 or the sequence illustrated in SEQID NO: 3. Particularly preferred substantially homologous glucoamylasesequences are the mature protein sequences as shown in FIG. 15 and whichcorrespond to SEQ ID NOs: 17, 18, 19, 20, 21, 22, 43, 44, 45, 46 and 47.Additionally, preferred substantially homologous glucoamylase sequencesare the sequences shown in FIG. 15, which correspond to SEQ ID NOs: 6,8, 10, 12, 14, 16, 29, 31, 33, 35 and 37 and include a leader sequence.Further substantially homologous polypeptides include allelic variationsand natural mutants having glucoamylase activity.

The glucoamylases of the present invention including substantiallyhomologous polypeptides and biologically functional fragments, have atleast 20%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% and at least 100% of theglucoamylase activity of the mature protein derived from Trichodermareesei having the sequence illustrated in FIG. 3 (SEQ ID NO: 4). In somepreferred embodiments of the invention, the specific activity of theglucoamylases tested under essentially the same conditions will be atleast 90%, at least 100%, at least 125%, at least 150%, at least 175%and also at least 200% of the specific activity of the mature proteinderived from Trichoderma reesei having the sequence illustrated in FIG.3 (SEQ ID NO: 4). In some embodiments, the specific activity may bemeasured on a soluble starch substrate and in other embodiments thespecific activity may be measured on a granular starch substrate.

In some embodiments, an amino acid sequence having at least 80% sequenceidentity to the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 will includeconservative amino acid substitutions using L-amino acids, wherein oneamino acid is replaced by another biologically similar amino acid.Conservative amino acid substitutions are those that preserve thegeneral charge, hydrophobicity/hydrophilicity, and/or steric bulk of theamino acid being substituted. Non-limiting examples of conservativesubstitutions include those between the following groups: Gly/Ala,Val/Ile/Leu, Lys/Arg, Asn/Gln, Glu/Asp, Ser/Cys/Thr and Phe/Trp/Tyr.Other conservative substitutions can be taken from the table below.

TABLE 1 Conservative Amino Acid Replacements For Amino Acid Code Replacewith any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine RD-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn,D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln AsparticAcid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys,S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu,D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp Isoleucine I D-Ile,Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu,D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His,D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

In other embodiments, the amino acid substitutions will not beconservative substitutions.

In some embodiments, it is contemplated that a glucoamylase of theinvention will be derived from a filamentous fungal strain andparticularly substantially homologous sequences will be obtained fromstrains of the genus Aspergillus spp., Rhizopus spp., Humicola spp.,Fusarium spp., Mucor spp., Trichoderma spp., and the like. In apreferred embodiment, substantially homologous sequences havingglucoamylase activity will be derived from strains of theTrichoderma/Hypocrea family cluster. Some of these species include T.stromaticum, H. citrina var. americana, H. citrina, H. lactea, H. hunua,T. fertile, T. tomentosum, H. vinosa, T. harzianum, T. inhamatum, T.oblongisporum, T. cf. aureoviride, T. cf. harzianum, T. fasciculatum, H.tawa, T. crassum, T. flavovirens, T. virens, T. longipilis, T. spirale,T. strictipilis, H. pilulifera, T. polysporum, T. croceum, T.minutisporum, T. hamatum, T. asperellum, T. atroviride, T. koningii, T.viride, H. gelatinosa, T. strigosum, T. pubescens, H. novazelandiae, T.saturnisporum, T. longibrachiatum, H. orientalis, T. citrinoviride, T.reesei, T. ghanense, T. pseudokonimgii, H. andinensis and H.aureoviride. Particularly preferred strains of the genus Trichoderma andallied Hypocrea spp. include H. citrina var. americana, H. citrina, H.lactea, H. vinosa, T. harzianum, T. atroviride, T. koningii, T. viride,H. gelatinosa, T. saturnisporum, T. longibrachiatum, H. orientalis, T.citrinoviride, T. reesei, and T. konilangbra.

Some strains of the species described above are accessible to the publicfrom culture collections such as American Type Culture Collection (ATCC)P. O. Box 1549, Manassas, Va. 20108; Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSM); Agricultural ResearchService Plant Culture Collection, Northern Regional Research Center(NRRL); the Centraalbureau voor Schimmelcultures (CBS), P. O. Box 85167,3508 AD Utrecht, The Netherlands; Plant Research Institute, Departmentof Agriculture, Mycology, Ottawa, (DAOM) Canada and InternationalMycological Institute (IMI), Genetic Resources Collection, Egham, UnitedKingdom.

Biologically Functional Glucoamylase Fragments

In some embodiments, the invention is related to biologically functionalfragments of the glucoamylase disclosed in SEQ ID NO: 3, SEQ ID NO: 4 orsubstantially homologous sequences thereto. In some embodiments, thebiologically functional fragment will include the catalytic domain of aglucoamylase encompassed by the invention. In other embodiments, thebiologically functional fragments will include at least 400 amino acidresidues, at least 425 amino acid residues, at least 450 amino acidresidues, and also at least 460 amino acid residues.

In some preferred embodiments, the fragment will encompass at least apart of the amino acid sequence represented by residue positions 1 to453 of SEQ ID NO: 4, and in other embodiments, the fragment willencompass positions 1 to 453 of SEQ ID NO: 4. In further preferredembodiments, the fragment will encompass the amino acid sequencerepresented by residue positions 1 to 453 of SEQ ID NO: 17; residuepositions 1 to 452 of SEQ ID NO: 18; residue positions 1 to 454 of SEQID NO: 19; residue positions 1 to 452 of SEQ ID NO: 20; residuepositions 1 to 453 of SEQ ID NO: 21; residue positions 1 to 453 of SEQID NO: 22; residue positions 1 to 452 of SEQ ID NO: 43; residuepositions 1 to 452 of SEQ ID NO: 44; residue positions 1 to 453 of SEQID NO: 45; residue positions 1 to 452 of SEQ ID NO: 46; or residuepositions 1 to 453 of SEQ ID NO: 47.

Biologically functional glucoamylase fragments encompassed by theinvention can be generated by method known in the art.

Glucoamylases having at least 85%, at least 90%, at least 93%, at least95%, at least 97%, at least 98% and at least 99% sequence identity tothe fragment which consists of amino acid residue 1 to 453 of SEQ ID NO:4 are also contemplated by the invention.

In other embodiments, the biologically functional fragments will includethe catalytic domain and the linker sequence of the glucoamylasedisclosed in SEQ ID NO: 4.

The biologically functional fragments may also comprise fusedpolypeptides or cleavable fused polypeptides in which anotherpolypeptide is fused at the N-terminus and/or the C-terminus of thepolypeptide. Techniques for producing fusion polypeptides are known inthe art.

Cloned Trichoderma Reesei and Substantially Homologous DNA Sequences

The invention also relates to a cloned DNA sequence coding for apolypeptide exhibiting glucoamylase activity of the invention, said DNAsequence comprising

-   -   a) the DNA sequence illustrated in SEQ ID NO: 1;    -   b) the DNA sequence illustrated in SEQ ID NO: 2;    -   c) a DNA sequence encoding a glucoamylase having at least 80%,        at least 83%, at least 85%, at least 87%, at least 90%, at least        93%, at least 95%, at least 97%, at least 98% and at least 99%        identity with the sequence of SEQ ID NO: 3;    -   d) a DNA sequence encoding a glucoamylase having at least 80%,        at least 83%, at least 85%, at least 87%, at least 90%, at least        93%, at least 95%, at least 97%, at least 98% and at least 99%        identity with the sequence of SEQ ID NO: 4;    -   e) a DNA sequence encoding an enzyme having glucoamylase        activity, wherein the enzyme has at least 95%, at least 96%, at        least 97%, at least 98% and at least 99% sequence identity to        any one of the sequences shown in SEQ ID NOs: 17, 18, 19, 20,        21, 22, 43, 44, 45, 46 and 47;    -   f) a DNA sequence encoding a biologically functional fragment of        a sequence having at least 85%, at least 90%, at least 95%, at        least 96%, at least 97% and at least 98% identity to amino acid        residue position 1 to 453 of the sequence shown in SEQ ID NO: 4;    -   g) a DNA sequence encoding an enzyme having glucoamylase        activity comprising an amino acid sequence having at least 90%,        at least 95%, at least 97% and at least 98% sequence identity to        any one of the following sequences        -   a. amino acid residue positions 1 to 453 of SEQ ID NO: 17;        -   b. amino acid residue positions 1 to 452 of SEQ ID NO: 18;        -   c. amino acid residue positions 1 to 454 of SEQ ID NO: 19;        -   d. amino acid residue positions 1 to 452 of SEQ ID NO: 20;        -   e. amino acid residue positions 1 to 453 of SEQ ID NO: 21;        -   f. amino acid residue positions 1 to 453 of SEQ ID NO: 22;        -   g. amino acid residue positions 1 to 452 of SEQ ID NO: 43;        -   h. amino acid residue positions 1 to 452 of SEQ ID NO: 44;        -   i. amino acid residue positions 1 to 453 of SEQ ID NO: 45;        -   j. amino acid residue positions 1 to 452 of SEQ ID NO: 46;            and        -   k. amino acid residue positions 1 to 453 of SEQ ID NO: 47.    -   h) a DNA which is at least 80%, at least 85%, at least 90%, at        least 93%, at least 95%, at least 97% and at least 99% identical        to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein        said DNA sequence codes for an enzyme having glucoamylase        activity; or    -   i) a DNA sequence, which hybridizes under high stringent        conditions to a nucleic acid probe corresponding to the DNA        sequence of SEQ ID NO: 2 or a fragment thereof having at least        20, at least 30 at least 40, at least 50 at least 60, at least        70 at least 100, at least 150 consecutive nucleotides.

The invention additionally encompasses a cloned DNA sequence encoding anenzyme having glucoamylase activity and at least 95%, at least 96%, atleast 97% at least 98% and at least 99% sequence identity to the aminoacid sequences of any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 29, 31,33, 35, and 37.

Because of the degeneracy of the genetic code, more than one codon maybe used to code for a particular amino acid. Therefore, different DNAsequences may encode a polypeptide having exactly the same amino acidsequence as the polypeptide of, for example SEQ ID NO: 4. The presentinvention encompasses polynucleotides, which encode the samepolypeptide. DNA sequences, which encode glucoamylases encompassed bythe invention may or may not include introns.

Homology of DNA sequences is determined by the degree of identitybetween two DNA sequences. Homology may be determined using computerprograms as described above for determining protein sequence homology.

A nucleic acid is hybridizable to another nucleic acid when a singlestranded form of the nucleic acid can anneal to the other nucleic acidunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known in the art forhybridization under low, medium, medium/high, high and very highstringency conditions (See., e.g. Sambrook et al., supra, particularlychapters 9 and 11). In general, hybridization involves a nucleotideprobe and a homologous DNA sequence that form stable double strandedhybrids by extensive base-pairing of complementary polynucleotides (See,Chapter 8, GeneCloning, An Introduction, T. A. Brown, (1995) Chapman andHall, London).

The filter with the probe and homologous sequence are washed in 2×sodium chloride/sodium citrate (SSC), 0.5% SDS at about 60° C. (mediumstringency); 65° C. (medium/high stringency) 70° C. (high stringency)and about 75° C. (very high stringency).

Vectors

According to one embodiment of the invention, a DNA construct comprisinga nucleic acid sequence encoding a glucoamylase encompassed by theinvention and operably linked to a promoter sequence is assembled totransfer into a host cell. The DNA construct may be introduced into ahost cell using a vector. The vector may be any vector which whenintroduced into a host cell is integrated into the host cell genome andis replicated. Vectors include cloning vectors, expression vectors,shuttle vectors, plasmids, phage particles, cassettes and the like. Insome preferred embodiments, the vector is an expression vector thatcomprises regulatory sequences operably linked to the glucoamylasecoding sequence.

Examples of suitable expression and/or integration vectors are providedin Sambrook et al., (1989) supra, and Ausubel (1987) supra, and van denHondel et al. (1991) in Bennett and Lasure (Eds.) MORE GENEMANIPULATIONS IN FUNGI, Academic Press pp. 396-428 and U.S. Pat. No.5,874,276. Reference is also made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, <www.fgsc.net>) for a list of vectors.Particularly useful vectors include vectors obtained from for examplesInvitrogen and Promega. Specific vectors suitable for use in fungal hostcells include vectors such as pFB6, pBR322, pUC18, pUC100, PDON™201,PDONR™221, PENTR™, pGEM®3Z and pGEM®4Z.

In some preferred embodiments, the promoter, which shows transcriptionalactivity in a fungal host cell may be derived from genes encodingproteins either homologous or heterologous to the host cell. Thepromoter may be a mutant, truncated and hybrid promoter. Preferably, thepromoter is useful in a Trichoderma or Aspergillus host. Exemplarypromoters include the T. reesei promoters cbh1, cbh2, egl1, egl2, eg5,xln1 and xln2. Other examples of useful promoters include promoters fromA. awamori and A. niger glucoamylase genes (glaA) (See, Nunberg et al.,(1984) Mol. Cell Biol. 4:2306-2315 and Boel et al., (1984) EMBO J.3:1581-1585), Aspergillus nidulans acetamidase genes and Rhizomucormiehei lipase genes.

In one embodiment, the promoter is one that is native to the host cell.For example, when T. reesei is the host, the promoter is a native T.reesei promoter.

In another embodiment, the promoter is one that is heterologous to thefungal host cell.

In a preferred embodiment, the promoter is T. reesei cbh1, which is aninducible promoter and has been deposited in GenBank under Accession No.D86235.

An “inducible promoter” is a promoter that is active under environmentalor developmental regulation. In some embodiments, the DNA constructincludes nucleic acids coding for a signal sequence that is an aminoacid sequence linked to the amino terminus of the polypeptide whichdirects the encoded polypeptide into the cell's secretory pathway. The5′ end of the coding sequence of the nucleic acid sequence may naturallyinclude a signal peptide coding region which is naturally linked intranslation reading frame with the segment of the glucoamylase codingsequence which encodes the secreted glucoamylase or the 5′ end of thecoding sequence of the nucleic acid sequence may include a signalpeptide which is foreign to the coding sequence. In some preferredembodiments, the DNA construct includes a signal sequence that isnaturally associated with the glucoamylase gene to be expressed.Effective signal sequences may include the signal sequences obtainedfrom glucoamylases of other filamentous fungal cells, such as fromHumicola, Aspergillus, and Rhizopus.

In preferred embodiments, the nucleic acid of the DNA construct codesfor a signal sequence having at least 95%, at least 96%, at least 97%,at least 98% and at least 99% sequence identity to the signal sequencedepicted in FIG. 3.

In additional embodiments, a DNA construct or vector comprising a signalsequence and a promoter sequence to be introduced into a fungal hostcell are derived from the same source. For example, in some embodiments,the signal sequence is the cbhl signal sequence which is operably linkedto a cbhl promoter. In other preferred embodiments the nativeglucoamylase signal sequence of a Trichoderma/Hypocrea family clustermember will be used.

In some embodiments, the expression vector also includes a terminationsequence. Any terminator sequence functional in the host cell may beused in the present invention. In one embodiment, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is homologous to the hostcell. A particularly suitable terminator sequence is cbhl derived from aTrichoderma strain and particularly T. reesei. Other useful fungalterminators include the terminator from A. niger or A. awamoriglucoamylase genes (Nunberg et al. (1984) supra, and Boel et al., (1984)supra), Aspergillus nidulans anthranilate synthase genes, Aspergillusoryzae TAKA amylase genes, or A. nidulans trpC (Punt et al., (1987) Gene56:117-124).

In some embodiments, an expression vector includes a selectable marker.Examples of preferred selectable markers include ones which conferantimicrobial resistance (e.g., hygromycin and phleomycin). Nutritionalselective markers also find use in the present invention including thosemarkers known in the art as amdS, argB and pyr4. Markers useful invector systems for transformation of Trichoderma are known in the art(See, e.g., Finkelstein, chapter 6 in BIOTECHNOLOGY OF FILAMENTOUSFUNGI, Finkelstein et al. Eds. Butterworth-Heinemann, Boston, Mass.(1992), Chap. 6.; and Kinghorn et al. (1992) APPLIED MOLECULAR GENETICSOF FILAMENTOUS FUNGI, Blackie Academic and Professional, Chapman andHall, London). In a preferred embodiment, the selective marker is theamds gene, which encodes the enzyme acetamidase, allowing transformedcells to grow on acetamide as a nitrogen source. The use of A. nidulansamds gene as a selective marker is described in Kelley et al., (1985)EMBO J. 4:475-479 and Penttila et al., (1987) Gene 61:155-164.

Methods used to ligate the DNA construct comprising a nucleic acidsequence encoding a glucoamylase, a promoter, a terminator and othersequences and to insert them into a suitable vector are well known inthe art. Linking is generally accomplished by ligation at convenientrestriction sites. If such sites do not exist, synthetic oligonucleotidelinkers are used in accordance with conventional practice. (See,Sambrook (1989) supra, and Bennett and Lasure, MORE GENE MANIPULATIONSIN FUNGI, Academic Press, San Diego (1991) pp 70-76.). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

Host Cells

The present invention also relates to host cells comprising a nucleicacid sequence encoding a glucoamylase of the invention, which are usedin the production of the glucoamylases of the invention. Preferred hostcells according to the invention are filamentous fungal cells, and theterm host cell includes both the cells, progeny of the cells andprotoplasts created from the cells of a filamentous fungal strains.

The term “filamentous fungi” refers to all filamentous forms of thesubdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORYMYCOLOGY, Wiley, N.Y.). These fungi are characterized by a vegetativemycelium with a cell wall composed of chitin, cellulose, and othercomplex polysaccharides. The filamentous fungi of the present inventionare morphologically, physiologically, and genetically distinct fromyeasts. Vegetative growth by filamentous fungi is by hyphal elongationand carbon catabolism is obligatory aerobic. In the present invention,the filamentous fungal parent cell may be a cell of a species of, butnot limited to, Trichoderma, (e.g., Trichoderma reesei, the asexualmorph of Hypocrea jecorina, T. Iongibrachiatum, Trichoderma viride,Trichoderma koningii, Trichoderma harzianum); Penicillium sp., Humicolasp. (e.g., H. insolens, H. lanuginosa and H. grisea); Chrysosporium sp.(e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A.oryzae, A. niger, A. nidulans, and A. awamori), Fusarium sp.,(e.g. F.graminum and F venenatum), Neurospora sp., Hypocrea sp., Mucor, andEmericella sp. (See also, Innis et al., (1985) Sci. 228:21-26). The term“Trichoderma” or “Trichoderma sp.” refer to any fungal genus previouslyor currently classified as Trichoderma. In some embodiments, the hostcell will be a genetically engineered host cell wherein native geneshave been inactivated, for example by deletion. Where it is desired toobtain a fungal host cell having one or more inactivated genes knownmethods may be used (e.g. methods disclosed in U.S. Pat. No. 5,246,853,U.S. Pat. No. 5,475,101 and WO92/06209). Gene inactivation may beaccomplished by complete or partial deletion, by insertionalinactivation or by any other means which renders a gene nonfunctionalfor its intended purpose (such that the gene is prevented fromexpression of a functional protein). Any gene from a Trichoderma sp. orother filamentous fungal host, which has been cloned can be deleted. Insome preferred embodiments, when the host cell is a Trichoderma cell andparticularly a T. reesei host cells the cbh1, cbh2, egl1 and egl2 geneswill be inactivated and preferably deleted. Particualrly preferredTrichoderma reesei host cells having quad-deleted proteins are set forthand described in U.S. Pat. No. 5,847,276 and WO 05/001036.

Transformation of Host Cells

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, (e.g., lipofection mediatedand DEAE-Dextrin mediated transfection); incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art (See, e.g., Ausubel et al., (1987),supra, chapter 9; and Sambrook (1989) supra, and Campbell et al., (1989)Curr. Genet. 16:53-56). The expression of heterologous protein inTrichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No.6,268,328; Harkki et al. (1991); Enzyme Microb. Technol. 13:227-233;Harkki et al., (1989) Bio Technol. 7:596-603; EP 244,234; EP 215,594;and Nevalainen et al., “The Molecular Biology of Trichoderma and itsApplication to the Expression of Both Homologous and HeterologousGenes”, in MOLECULAR INDUSTRIAL MYCOLOGY, Eds. Leong and Berka, MarcelDekker Inc., NY (1992) pp. 129-148). Reference is also made to Cao etal., (2000) Sci. 9:991-1001 and EP 238 023 for transformation ofAspergillus strains and WO96/00787 for transformation of Fusariumstrains.

Preferably, genetically stable transformants are constructed with vectorsystems whereby the nucleic acid encoding the glucoamylase is stablyintegrated into a host strain chromosome. Transformants are thenpurified by known techniques. In one nonlimiting example, stabletransformants including an amdS marker are distinguished from unstabletransformants by their faster growth rate and the formation of circularcolonies with a smooth, rather than ragged outline on solid culturemedium containing acetamide. Additionally, in some cases a further testof stability is conducted by growing the transformants on solidnon-selective medium (ie., NH₄(SO₄)₂ (5 mg/mL) as a nitrogen source),harvesting spores from this culture medium and determining thepercentage of these spores which subsequently germinate and grow onselective medium containing 10 mM acetamide as a sole nitrogen source.Alternatively, other methods known in the art may be used to selecttransformants.

In one specific embodiment, the preparation of Trichoderma sp. fortransformation involves the preparation of protoplasts from fungalmycelia (See, Campbell et al., (1989) Curr. Genet 16:53-56). Alsoagrobacterium tumefaciens-mediated transformation of filamentous fungiis known (See, de Groot et al., (1998) Nat. Biotechnol. 16:839-842).

In some embodiments, the mycelia are obtained from germinated vegetativespores. The mycelia are treated with an enzyme that digests the cellwall resulting in protoplasts. The protoplasts are then protected by thepresence of an osmotic stabilizer in the suspending medium. Thesestabilizers include sorbitol, mannitol, potassium chloride, magnesiumsulfate and the like. Usually the concentration of these stabilizersvaries between 0.8 M and 1.2 M. It is preferable to use about a 1.2 Msolution of sorbitol in the suspension medium. Uptake of DNA into thehost Trichoderma sp. strain is dependent upon the calcium ionconcentration. Generally, between about 10 mM CaCl₂ and 50 mM CaCl₂ isused in an uptake solution. Reference is also made to U.S. Pat. No.6,022,725 and U.S. Pat. No. 6,268,328 for transformation procedures usedwith filamentous fungal hosts.

The present invention relates to methods of recombinantly producing theglucoamylase comprising expressing a polynucleotide encoding aglucoamylase of the invention in a filamentous fungal host cell andcultivating the host cell under conditions suitable for production ofthe glucoamylase and optionally recovering the glucoamylase.

In the expression and production methods of the present invention thefungal cells are cultured under suitable conditions in shake flaskcultivation, small scale or large scale fermentations (includingcontinuous, batch and fed batch fermentations ) in laboratory orindustrial fermentors, with suitable medium containing physiologicalsalts and nutrients (See, e.g., Pourquie, J. et al., BIOCHEMISTRY ANDGENETICS OF CELLULOSE DEGRADATION, eds. Aubert, J. P. et al., AcademicPress, pp. 71-86, 1988 and Ilmen, M. et al., (1997) Appl. Environ.Microbiol. 63:1298-1306). Common commercially prepared media (e.g.,Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth and SabouraudDextrose (SD) broth) find use in the present invention. Preferredculture conditions for a given filamentous fungus are known in the artand may be found in the scientific literature and/or from the source ofthe fungi such as the American Type Culture Collection and FungalGenetics Stock Center. In cases where a glucoamylase coding sequence isunder the control of an inducible promoter, the inducing agent (e.g., asugar, metal salt or antimicrobial), is added to the medium at aconcentration effective to induce glucoamylase expression.

In some embodiments, in order to evaluate the expression of aglucoamylase by a cell line that has been transformed with apolynucleotide encoding a glucoamylase encompassed by the invention,assays are carried out at the protein level, the RNA level and/or by useof functional bioassays particular to glucoamylase activity and/orproduction. Some of these assays include Northern blotting, dot blotting(DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chainreaction), or in situ hybridization, using an appropriately labeledprobe (based on the nucleic acid coding sequence) and conventionalSouthern blotting and autoradiography.

In addition, the production and/or expression of a glucoamylase may bemeasured in a sample directly, for example, by assays directly measuringreducing sugars such as glucose in the culture medium and by assays formeasuring glucoamylase activity, expression and/or production. Inparticular glucoamylase activity may be assayed by the3,5-dinitrosalicylic acid (DNS) method (See, Goto et al., (1994) Biosci.Biotechnol. Biochem. 58:49-54). In additional embodiments, proteinexpression, is evaluated by immunological methods, such asimmunohistochemical staining of cells, tissue sections or immunoassay oftissue culture medium, (e.g., by Western blot or ELISA). Suchimmunoassays can be used to qualitatively and quantitatively evaluateexpression of a glucoamylase. The details of such methods are known tothose of skill in the art and many reagents for practicing such methodsare commercially available.

The glucoamylases of the present invention may be recovered or purifiedfrom culture media by a variety of procedures known in the art includingcentrifugation, filtration, extraction, precipitation and the like.

Uses and Compositions

The present invention is also directed to compositions comprisingglucoamylases of the invention and methods of using the glucoamylases inindustrial and commercial applications. Nonlimiting examples, whichinclude the use of glucoamylases encompassed by the invention inindustrial and commercial applications are briefly described below.

The glucoamylases may be used in starch hydrolyzing and saccharifyingcompositions, cleaning and detergent compositions (e.g., laundrydetergents, dish washing detergents, and hard surface cleaningcompositions), and in animal feed compositions. Further theglucoamylases may be used in baking applications, such as bread and cakeproduction, brewing, healthcare, textile, environmental waste conversionprocesses, biopulp processing, and biomass conversion applications.

In particular, the glucoamylases may be used for starch conversionprocesses, and particularly in the production of dextrose for fructosesyrups, specialty sugars and in alcohol and other end-product (e.g.organic acid, ascorbic acid, and amino acids) production fromfermentation of starch containing substrates (G. M. A van Beynum et al.,Eds. (1985) STARCH CONVERSION TECHNOLOGY, Marcel Dekker Inc. NY).Dextrins produced using glucoamylase compositions of the invention mayresult in glucose yields of at least 80%, at least 85%, at least 90% andat least 95%. Production of alcohol from the fermentation of starchsubstrates using glucoamylases encompassed by the invention may includethe production of fuel alcohol or portable alcohol.

In one preferred embodiment, the glucoamylases of the invention willfind use in the hydrolysis of starch from various plant-basedsubstrates, which are used for alcohol production. In some preferredembodiments, the plant-based substrates will include corn, wheat,barley, rye, milo, rice, sugar cane and combinations thereof. In someembodiments, the plant-based substrate will be fractionated plantmaterial, for example a cereal grain such as corn, which is fractionatedinto components such as fiber, germ, protein and starch (endosperm)(U.S. Pat. No. 6,254,914 and U.S. Pat. No. 6,899,910). Methods ofalcohol fermentations are described in THE ALCOHOL TEXTBOOK, A REFERENCEFOR THE BEVERAGE, FUEL AND INDUSTRIAL ALCOHOL INDUSTRIES, 3^(rd) Ed.,Eds K. A. Jacques et al., 1999, Nottingham University Press, UK. Incertain preferred embodiments, the alcohol will be ethanol. Inparticular, alcohol fermentation production processes are characterizedas wet milling or dry milling processes. In some embodiments, theglucoamylase will be used in a wet milling fermentation process and inother embodiments the glucoamylase will find use in a dry millingprocess.

Dry grain milling involves a number of basic steps, which generallyinclude: grinding, cooking, liquefaction, saccharification, fermentationand separation of liquid and solids to produce alcohol and otherco-products. Plant material and particularly whole cereal grains, suchas corn, wheat or rye are ground. In some cases the grain may be firstfractionated into component parts. The ground plant material may bemilled to obtain a coarse or fine particle. The ground plant material ismixed with liquid in a slurry tank. The slurry is subjected to hightemperatures in a jet cooker along with liquefying enzymes (e.g. alphaamylases) to solubles and hydrolyze the starch in the cereal todextrins. The mixture is cooled down and further treated withsaccharifying enzymes, such as glucoamylases encompassed by the instantinvention, to produce glucose. The mash containing glucose is thenfermented for approximately 24 to 120 hours in the presence offermentation microorganisms, such as ethanol producing microorganism andparticularly yeast (Saccharomyces spp). The solids in the mash areseparated from the liquid phase and alcohol such as ethanol and usefulco-products such as distillers' grains are obtained.

In some embodiments, the saccharification step and fermentation step arecombined and the process is referred to as simultaneous saccharificationand fermentation or simultaneous saccharification, yeast propagation andfermentation.

In other embodiments, the cooking step or exposure of the starchcontaining substrate to temperatures above the gelatinization temperateof the starch in the substrate may be eliminated. These fermentationprocesses in some embodiments include milling of a cereal grain orfractionated grain and combining the ground cereal grain with liquid toform a slurry which is then mixed in a single vessel with a glucoamylaseaccording to the invention and optionally other enzymes such as but notlimited to alpha amylases, other glucoamylases and enzymes havinggranular starch hydrolyzing activity and yeast to produce ethanol andother co-products (U.S. Pat. No. 4,514,496, WO 04/081193 and WO04/080923).

In some embodiments, the invention pertains to a method of saccharifyinga liquid starch solution, which comprises an enzymatic saccharificationstep using a glucoamylase of the invention.

In some embodiments, an enzyme composition including a glucoamylaseencompassed by the invention and obtained in culture media or recoveredand purified from the culture medium will be optionally used incombination with any one or combination of the following enzymes—alphaamylases, proteases, pullulanases, isoamylases, cellulases,hemicellulases, xylanases, cyclodextrin glycotransferases, lipases,phytases, laccases, oxidases, esterases, cutinases, xylanases, granularstarch hydrolyzing enzyme and other glucoamylases.

In some particularly preferred compositions the glucoamylases of theinvention will be combined with alpha amylases, such as fungal alphaamylases (e.g. Aspergillus sp.) or bacterial alpha amylases (e.g.Bacillus sp. such as B. stearothermophilus, B. amyloliquefaciens and B.licheniformis) and variants thereof. In some embodiments the alphaamylase will be an alpha amylase having at least 90%, 93%, 95%, 96%,97%, 98% and 99% sequence identity to the mature protein sequence of SEQID NO: 27. Commercially available alpha amylases contemplated for use inthe compositions of the invention are known and include GZYME G997,SPEZYME FRED, SPEZYME EHTYL (Genencor International Inc.) and TERMAMYL120-L and SUPRA (Novozymes, Biotech.).

In other particularly preferred embodiments, the glucoamylases of theinvention will be combined with other glucoamylases. In someembodiments, the glucoamylases of the invention will be combined withone or more glucoamylases derived from strains of Aspergillus orvariants thereof, such as A. oryzae, A. niger (e.g., the mature proteinsequence of FIG. 20(A), A. kawachi, and A. awamori; glucoamylasesderived from strains of Humicola or variants thereof, particualrly H.grisea, such as the glucoamylase having at least 90%, 93%, 95%, 96%,97%, 98% and 99% sequence identity to SEQ ID NO: 3 disclosed in WO05/052148; glucoamylases derived from strains of Talaromyces or variantsthereof, particularly T. emersonii; and glucoamylases derived fromstrains of Athelia and particularly A. rolfsii.

Material and Methods

In the disclosure and experimental section which follows, the followingabbreviations apply:

TrGA (a Trichoderma reesei glucoamylase composition, the mature proteinhaving the amino acid sequence of SEQ ID NO: 4); AkAA (an Aspergilluskawachi alpha amylase composition having the mature protein of sequenceSEQ ID NO: 27); AnGA (DISTILLASE comprising an Aspergillus niger GA(Genencor International Inc.,)); GA (glucoamylase); GAU (glucoamylaseunit); AAU (alpha amylase unit); wt % (weight percent); ° C. (degreesCentigrade); rpm (revolutions per minute); H₂O (water); dH₂O (deionizedwater); dIH₂O (deionized water, Milli-Q filtration); aa or AA (aminoacid); bp (base pair); kb (kilobase pair); kD or kDa (kilodaltons); g orgm (grams); pg (micrograms); mg (milligrams); pL (microliters); ml andmL (milliliters); mm (millimeters); μm (micrometer); M (molar); mM(millimolar); pM (micromolar); U (units); V (volts); MW (molecularweight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); DO(dissolved oxygen); and EtOH (ethanol).

The following assays and methods are used in the examples providedbelow:

1) GA Assay—Glucoamylase assay: Glucoamylase activity was measure usinga well-known assay which is based on the ability of glucoamylase tocatalyze the hydrolysis of p-nitrophenyl-alpha-D-glucopyranoside (PNPG)to glucose and p-nitrophenol. At an alkaline pH, the nitrophenol forms ayellow color that is proportional to glucoamylase activity and ismonitored at 400 nm and compared against an enzyme standard measured asa GAU (Elder, M. T. and Montgomery R. S., Glucoamylase activity inindustrial enzyme preparations using colorimetric enzymatic method,Journal of AOAC International, vol. 78(2), 1995).

One GAU is defined as the amount of enzyme that will produce 1 gm ofreducing sugar calculated as glucose per hour from a soluble starchsubstrate (4% ds) at pH 4.2 and 60° C.

2) Primers and PCR Protocol for Amplification of Genes fromTrichoderma/Hypocrea Strains:

Trichoderma/ Hypocrea SEQ ID GA-gene Primer Gene Specific Sequence NO:Trichoderma NSP231F ATGCCCGCCTTCGCCATGGACC 23 reesei NSP232RTTACGACTGCCAGGTGTCCTCC 24 NSP233F ATGCACGTCCTGTCGACTGCGG 25

Component μl Forward primer (10 μM) 1 Reverse primer (10 μM) 1 Templategenomic DNA 5 dNTP (10 mM) 1 HiFi Buffer 5 MgSO₄ (50 mM) 2 DNApolymerase - Platinum 0.5 Taq Polymerase High Fidelity (Invitrogen, cat.No. 11304- 029 Milli-Q water, sterile 34.5

With respect to the PCR program, initial denaturation was 2 min, at 94°C. for 1 cycle; denaturation 30 sec, at 94° C., annealing for 30 sec, at55° C. and extension for 2 min at 68° C. for 30 cycles and a finalextension step of 7 min at 68° C.

3) Ethanol and Carbohydrate Determinations

Ethanol and carbohydrate composition of the samples were determinedusing the HPLC method as described herein:

-   a) a 1.5 mL Eppendorf centrifuge tube was filled with fermentor beer    and cooled on ice for 10 min;-   b) the sample tube was centrifuged for 1 min in Eppendorf table top    centrifuge;-   c) a 0.5 mL sample of the supernatant was transferred to a test tube    containing 0.05 mL of Kill solution (1.1 N H₂SO₄) and allowed to    stand for 5 min;-   d) 5.0 mL of water is added to the test tube sample and then    filtered into a HPLC vial through 0.45 pm Nylon Syringe Filter; and-   e) run on HPLC.

HPLC conditions:

a) Ethanol System: Column: Phenomenex Rezex Organic Acid Column(RHM-Monosaccharide) #00H 0132-KO (Equivalent to Bio-Rad 87H); ColumnTemperature: 60° C.; Mobile Phase: 0.01 N H₂SO₄; Flow Rate: 0.6 mL/min;Detector: RI; and

b) Injection Volume: 20 μL.

c) Carbohydrate System: Column: Phenomenex Rezex Carbohydrate(RCM-Monosaccharide) #OOH-0130-KO (Equivalent to Bio-Rad 87H); ColumnTemperature: 70° C.; Mobile Phase: Nanopure DI H₂O; Flow Rate: 0.8mL/min; Detector: RI; Injection Volume: 10 μL (3% DS material)

The column separates based on the molecular weight of the saccharides,which are designated as DP-1 (monosaccharides); DP-2 (disaccharides);DP-3 (trisaccharides) and DP>3 (oligosaccharide sugars having a degreeof polymerization greater than 3).

4) Residual starch iodine test: A sample of the beer (fermentationbroth) was centrifuged in 2 ml plastic centrifuge tubes. The supernatantwas decanted and the tube containing the pellet was placed in an icebath. Several drops of 0.025N iodine solution (0.1 N iodine from VWRCat. No. VW3207-1 diluted 4×) was added to the pellet and mixed. Apositive (+) starch shows a range of color from blue to purple and theintensity of color is directly proportional to the concentration ofstarch. A negative result (−) remains yellowish.

5) Determination of total starch content: The enzyme-enzyme starchliquefaction and saccharification process (dual enzyme method) was usedto determine the total starch content. In a typical analysis, 2 g of thedry sample was taken in a 100 ml Kohlraucsh flask and 45 ml of MOPSbuffer, pH 7.0 was added. The slurry was well stirred for 30 min.SPEZYME FRED (1:50 diluted in water), 1.0 ml was added and heated toboiling for 3-5 min. The flask was placed in an autoclave maintained at121° C. for 15 min. After autoclaving the flask was placed in a waterbath at 95° C. and 1 ml of 1:50 dilutes SPEZYME FRED was added andincubated for 45 min. The pH was adjusted to pH 4.2 and the temperaturewas reduced to 60° C. This was followed by addition of 20 ml acetatebuffer, pH 4.2. Saccharification was carried out by adding 1.0 ml of1:100 diluted OPTIDEX L-400 (Glucoamylase from Genencor InternationalInc.) and the incubation was continued for 18 hr at 60° C. The enzymereaction was terminated by heating at 95° C. for 10 min. The total sugarcomposition was determined by HPLC analysis using glucose as a standard.The soluble starch hydrolysate from water extraction of a sample at roomtemperature without enzymatic treatment was subtracted from the totalsugar.

6) Total protein analysis: The total nitrogen (N) in the samplepreparations was determined using the Kjeldhal method (American Assoc.Cereal Chemists (AACC), (1983), Methods 22B608th Ed. St Paul, Minn.).Protein content was calculated by 6.25× total N.

Examples

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspect of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Isolation and Cloning of the TrGA

Chromosomal DNA of Trichoderma reesei QM6a was isolated from mycelialmass of a liquid culture in Potato Dextrose Broth (Difco™ Cat. No.254920) using the BIO101 Fast Prep® System according to the methoddescribed by the supplier (Qbiogene). The DNA was purified using a QuickSpin column (Qiagen art No. 28106). The glucoamylase gene was isolatedusing primers with GA-specific sequences, NSP232R (SEQ ID NO: 24) andNSP233F (SEQ ID NO: 25) designed according to the predicted nucleotidesequence in the Trichoderma reesei genome database of Department ofEnergy (DOE) Joint Genome Institute. The primers were flanked at the5′-end by Gateway® attB sequences (Invitrogen). T. reesei QM6achromosomal DNA was used as template.

The PCR mix contained the following components: Forward primer (10 μM) 4μL; Reverse primer (10 M) 4μL; template DNA (500 ng/μL) 1 μL; dNTPmix(10 mM) 2 μL; 10× Cx buffer 10 μL and PfuTurbo® Cx Hotstart DNApolymerase 0.5 μL (Stratagen Cat. No. 600410). Deionized water was addedup to a total volume of 100 μL.

The PCR protocol was as follows: Initial denaturation for 30 sec. at 98°C., denaturation, annealing and extension in 30 cycles of 10 sec at 98°C.; 30 sec at 68° C.; 45 sec at 72° C., respectively, and a finalextension step of 10 min at 72° C.

The PCR fragments were analyzed by electrophoresis in 1% agarose.Fragments of the expected size were isolated using the Gel-ExtractionPurification Kit (Qiagene Cat. no. 28706). The PCR fragments were clonedinto the Gateway® Entry vector pDONR201 and transformed into E. coliDH5alpha Max Efficiency cells (Invitrogen Cat.No. 18258012). Thenucleotide sequence of the inserted DNA was determined, from which thegenomic DNA sequence of the TrGA gene was deduced (FIG. 1 (SEQ ID NO:1)).

Example 2 Transformation of T. Reesei and Fermentation/Expression of theTrGA

Vector DNA containing the correct GA gene sequence was recombined intothe T. reesei expression vector pTrex3g (FIG. 17).

The vector pTrex3g is based on the E. coli vector pSL1 180 (PharmaciaInc., Piscataway, N.J.) which is a pUC118 phagemid based vector(Brosius, J. (1989), DNA 8:759) with an extended multiple cloning sitecontaining 64 hexamer restriction enzyme recognition sequences. It wasdesigned as a Gateway destination vector (Hartley et al., (2000) GenomeResearch 10:1788-1795) to allow insertion using Gateway technology(Invitrogen) of any desired open reading frame between the promoter andterminator regions of the T. reesei cbh1 gene. It also contains theAspergillus nidulans amds gene for use as a selective marker intransformation of T. reesei. The details of the pTrex3g vector are asfollows (FIG. 17A). The vector is 10.3 kb in size. Inserted into thepolylinker region of pSL1180 are the following segments of DNA: a) A 2.2bp segment of DNA from the promoter region of the T. reesei cbh1 gene;b) the 1.7 kb Gateway reading frame A cassette acquired from Invitrogenthat includes the attR1 and attR2 recombination sites at either endflanking the chloramphenicol resistance gene (CmR) and the ccdB gene; c)a 336 bp segment of DNA from the terminator region of the T. reesei cbh1gene; and d) a 2.7 kb fragment of DNA containing the Aspergillusnidulans amdS gene with its native promoter and terminator regions.

The expression vector containing the T. reesei GA gene, pNSP23 (FIG. 17)was transformed into a T. reesei host strain derived from RL-P37 (IA52)and having various gene deletions (Δcbh1, Δcbh2, Δeg1, Δeg2) usingparticle bombardment by the PDS-1000/Helium System (BioRad Cat. No.165-02257). The protocol is outlined below, and reference is also madeto examples 6 and 11 of WO 05/001036.

A suspension of spores (approximately 5×10⁸ spores/ml) from a quaddeleted strain of T. reesei was prepared. 100 ul-200 ul of sporesuspension was spread onto the center of plates of Minimal Medium (MM)acetamide medium. (MM acetamide medium had the following compositions:0.6 g/L acetamide; 1.68 g/LCsCl; 20 g/L glucose; 20 g/L KH₂PO₄; 0.6 g/LCaCl₂ 2H₂0; 1 ml/L 1000× trace elements solution; 20 g/L Noble agar; andpH 5.5. 1000× trace elements solution contained 5.0 g/L FeSO₄ 7H₂O; 1.6g/L MnSO₄; 1.4 g/L ZnSO₄ 7H₂O and 1.0 g/L CoCl₂ 6H₂O. The sporesuspension was allowed to dry on the surface of the MM acetamide medium.

Transformation followed the manufacturers instruction. Briefly, 60 mg ofM10 tungsten particles were placed in a microcentrifuge tube. 1 mL ofethanol was added and allowed to stand for 15 seconds. The particleswere centrifuged at 15,000 rpm for 15 seconds. The ethanol was removedand the particles were washed three times with sterile dH₂O before 250uL of 50% (v/v) sterile glycerol was added. 25 ul of tungsten particlesuspension was placed into a microtrifuge tube. While continuouslyvortexing, the following were added: 5 ul (100-200 ng/ul) of plasmidDNA, 25 ul of 2.5M CaCl₂ and 10 ul of 0.1 M spermidine. The particleswere centrifuged for 3 seconds. The supernatant was removed and theparticles were washed with 200 ul of 100% ethanol and centrifuged for 3seconds. The supernatant was removed, 24 uL 100% ethanol was added andmixed by pipetting, then 8 ul aliquots of particles were removed andplaced onto the center of macrocarrier disks that were held in adesiccator. Once the tungsten/DNA solution had dried the macrocarrierdisk was placed in the bombardment chamber along with the plate of MMacetamide with spores and the bombardment process was performedaccording to the manufacturers instructions. After bombardment of theplated spores with the tungsten/DNA particles, the plates were incubatedat 30° C. Transformed colonies were transferred to fresh plates of MMacetamide medium and incubated at 30° C.

Example 3 Demonstration of GA Activity from the Expressed TrGA inTransformed Cells

After 5 days of growth on MM acetamide plates transformants displayingstable morphology were inoculated into 250 ml shake flasks containing 30ml of Proflo medium. (Proflo medium contained: 30 g/L a-lactose; 6.5 g/L(NH₄)₂SO₄; 2 g/L KH₂PO₄; 0.3 g/L MgSO₄ 7H₂O; 0.2 g/L CaCl₂; 1 ml/L 1000×trace element salt solution; 2 ml/L 10% Tween 80; 22.5 g/L ProFlocottonseed flour (Traders protein, Memphis, Tenn.); 0.72 g/L CaCO₃.After two days growth at 28 C and 140 rpm, 10% of the Proflo culture wastransferred to a 250 ml shake flask containing 30 ml of Lactose DefinedMedia. The composition of the Lactose defined Media was as follows 5 g/L(NH₄)₂SO₄; 33 g/L PIPPS buffers; 9 g/L casamino acids; 4.5 g/L KH₂PO₄;1.0 g/L MgSO₄ 7H₂O; 5 ml/L Mazu DF60-P antifoam (Mazur Chemicals, IL);1000× trace element solution; pH 5.5; 40 ml/L of 40% (w/v) lactosesolution was added to the medium after sterilization. The LactoseDefined medium shake flasks were incubated at 28° C., 140 rpm for 4-5days.

Mycelium was removed by centrifugation and the supernatant was analyzedfor total protein (BCA Protein Assay Kit, Pierce Cat. No. 23225) and GAactivity using pNPG as substrate (Sigma N-1377).

Samples of the culture supernatant were mixed with appropriate volume of2× sample loading buffer with reducing agent and the protein profile wasdetermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using NuPAGE® Novex 10% Bis-Tris Gel with MES SDS RunningBuffer. The gels were stained with SimplyBlue™ SafeStain (Invitrogen,Carlsbad, Calif., USA).

On SDS-PAGE analysis a protein band that was not observed in supernatantfrom a quad delete strain was observed in the supernatant of sometransformants with the pTrex3g vector containing the glucoamylase openreading frame (FIG. 16). This new protein band had an apparent molecularweight of approximately 64 kDa. This result confirms that TrGA issecreted into the medium.

Example 4 Biochemical Characterization of the GA Gene Product

GA producing transformants were grown at 4-L scale. The culture filtratewas concentrated using an ultra filtration unit with a nominal molecularweight limit of 10,000 Da (Pall Omega Centramate OS010c10). The crudeenzyme preparation was purified by a 2-step procedure using an AKTAexplorer 100 FPLC System (Amersham Biosciences). A HiPrep 16/10 FFQ-Sepharose column (Amersham BioSciences Cat. No. 17-5190-01) wasequilibrated with 25 mM Tris pH 8.0 and the protein was eluted from thecolumn with 100 mM NaCl in 25 mM Tris pH 8.0. A second affinitychromatography step was performed using Cbind 200 resin (Novagen Cat.No. 701212-3) and 50 mM Tris pH 7.0 containing 500 mM NaCl as elutionbuffer (FIG. 16). The N-terminus of the gene product(Ser-Val-Asp-Asp-Phe-Ile) (SEQ ID NO: 38) was determined by Edmandegradation (Edman, P. (1956) Acta Chem Scand 10:761-768).

The pH and temperature profiles of the glucoamylase activity of the geneproduct were determined using 4-nitrophenyl-α-D-glucopyranoside assubstrate (Elder, M. T. and Montgomery R. S., Glucoamylase activity inindustrial enzyme preparations using colorimetric enzymatic method;Collaborative study Journal of AOAC International, vol. 78(2), 1995)(FIG. 18).

Example 5 Isolation/Cloning of Glucoamylase Homologs from Strains in theTrichoderma/Hypocrea Family Cluster

Chromosomal DNA preparations of the strains (GA102)-Hypocrea citrinavar. americana (CBS976.69); (GA104)—Hypocrea vinosa (CBS960.68);(GA105)—Trichoderma sp; (GA107)—Hypocrea gelatinosa (CBS254.62);GA108—Hypocrea orientalis (ATCC 90550); (GA109)—Trichoderma konilangbra;(GA103)—Trichoderma harzianum (CBS433.95); (GA113)—Trichoderma sp.;(GA124)—Trichoderma longibrachiatum; (GA127)—Trichoderma asperellum(ATCC 28020); and (GA128)—Trichoderma strictipilis (CBS 347.93) wereisolated as described in example 1. Full-length GA genes were cloned asdescribed in example 1 using the TrGA-gene specific primers NSP231 F(SEQ ID NO: 23) and NSP232R (SEQ ID NO: 24). The nucleotide sequences ofthe strains are disclosed in FIG. 4 for GA102 (SEQ ID NO: 5); FIG. 5 forGA104 (SEQ ID NO: 7); FIG. 6 for GA105 (SEQ ID NO: 9); FIG. 7 for GA107(SEQ ID NO: 11); FIG. 8 for GA108 (SEQ ID NO: 13); FIG. 9 for GA109 (SEQID NO: 15); FIG. 10 for GA113 (SEQ ID NO: 28); FIG. 11 for GA103 (SEQ IDNO: 30); FIG. 12 for GA124 (SEQ ID NO: 32); FIG. 13 for GA127 (SEQ IDNO: 34) and FIG. 14 for GA128 (SEQ ID NO: 36). The corresponding aminoacid sequences are illustrated in FIG. 15. Table 2 sets forth thepercent identity of the amino acid sequences of the mature protein of T.reesei glucoamylase (FIG. 3B, SEQ ID NO: 4) with the glucoamylasehomologs from the Hypocrea/Trichoderma cluster.

TABLE 2 % Identity of GA homologs from the Hypocrea/Trichoderma clusterGA102 GA103 GA104 GA105 GA107 GA108 GA109 GA113 GA124 GA127 GA128 TrGAGA102 100 86 86 84 87 84 84 83 84 87 85 84 GA103 100 98 90 96 90 91 8690 98 90 90 GA104 100 91 97 91 90 86 91 99 90 91 GA105 100 90 95 93 8394 91 94 95 GA107 100 90 90 86 90 98 90 90 GA108 100 94 84 98 91 94 97GA109 100 83 94 91 94 94 GA113 100 84 86 83 84 GA124 100 91 94 98 GA127100 91 91 GA128 100 94 TrGA 100

T. reesei strains over-expressing GA were obtained as described inexample 2. Crude enzyme preparations were obtained as described inexample 3 and FIG. 19 illustrates the gels obtained for some of thehomologs. Table 3 sets forth the glucoamylase activity of some of thehomologs.

TABLE 3 Total protein U Specific Strain Gene from: mg/mL GA/mL ActivityGA104 H. vinosa 2.76 37 13 GA105 T. sp. 2.77 26 9 GA107 H. gelatinosa3.61 178 49 GA109 T. konilangbra 2.22 10 5 TrGA T. reesei 3.89 91 23Host T. reesei 0.7 3 4 Control

Example 6 Glucose Production Using TrGA

A 32% DS slurry of Cargill bag starch was made up with reverse osmosiswater. The pH of the slurry was adjusted to pH 5.8. The slurry wasfiltered through a 100-mesh screen and dosed at 4.0 AAU/g ds usingSPEZYME® ETHYL, (Genencor International, Inc.). The slurry was thenjetted at 107.3° C. for 5 min (primary liquefaction). Enzyme activity isdetermined by the rate of starch hydrolysis, as reflected in the rate ofdecrease in iodine-staining capacity. One AAU unit of bacterial alphaamylase activity is the amount of enzyme required to hydrolyze 10 mg ofstarch per minute under specified conditions. After primaryliquefaction, the liquefact was collected and placed in a 95° C. waterbath for 120 min (Secondary liquefaction). Samples were taken at 30, 60,90 and 120 min and checked for DE by using the standard Schoorlsreducing sugar method from the Corn Refiners Association. The liquefactwas aliquoted in 100-g quantities into screw cap jars, the pH wasadjusted to pH 4.5 and equilibrated to 60° C. for 15 minutes prior todosing. The TrGA enzyme was diluted so as to add 0.2 mis of dilutedenzyme to the jar at 0.22 GAU/g ds. After dosing, the liquefact wasaliquoted into 7 screw cap tubes, each containing approximately 10 mlsof material and returned to the designated temperature. Tubes wereremoved at selected time intervals (18, 24, 30, 42, 50 and 55 hours) andanalyzed by HPLC Carbohydrate System: Column: Phenomenex RezexCarbohydrate (RCM- Monosaccharide) #00H-0130-KO (Equivalent to Bio-Rad87H); Column Temperature: 70° C.; Mobile Phase: Nanopure DI H₂O; FlowRate: 0.8 mL/min; Detector: RI; Injection Volume: 10 uL (3% DS material)for sugar composition.

TABLE 4 Production of Glucose from cornstarch using TrGA Treatment (hrs)% DP1 % DP2 % DP3 % DP > 3 18 80.66 2.60 0.66 16.08 24 84.25 2.31 0.4612.98 30 86.26 2.66 0.47 10.61 42 88.85 3.05 0.40 7.69 50 89.93 3.260.42 6.39 55 90.64 3.36 0.37 5.62

Example 7 Ethanol Production Using TrGA in a SimultaneousSaccharification and Fermentation (SSF) Process

A sample of corn mash liquefact from a local ethanol producer wasobtained and diluted to 29% DS using thin stillage. The pH of the slurrywas adjusted to pH 4.3 using 6 N sulphuric acid. A 300 g aliquot of themash was placed into a 31° C. water bath and allowed to equilibrate.TrGA was added to the sample (0.4 GAU/g ds, which is equal to 1.08 kg/MTds). After enzyme addition, 1 ml of a 15 g in 45 ml DI water solution ofRed Star Red yeast (Lesaffre yeast Corp. Milwaukee, Wis.) was added toeach sample. Samples were taken at 18, 26, 41 and 53 hours and analyzedby HPLC Column: Phenomenex Rezex organic Acid Column(RHM-Monosaccharide) #00H-0132-KO (Equivalent to Bio-Rad 87H); ColumnTemperature: 60° C.; Mobile Phase: 0.01 NH₂SO₄; Flow Rate: 0.6 mL/min;Detector: RI; Injection Volume: 20 uL.

TABLE 5 Production of Ethanol by TrGA (0.4 GAU/g) in a SSF Process Sam-% w/v ple % w/v % w/v % w/v % w/v Lactic % w/v % v/v (hrs) DP > 3 DP-3DP-2 DP-1 Acid Glycerol EtOH 18 6.38 0.61 3.42 2.69 0.31 0.97 7.44 264.39 0.76 1.02 1.81 0.30 1.12 10.68 41 1.62 0.47 0.35 0.77 0.31 1.2713.65 53 1.03 0.37 0.36 0.16 0.32 1.32 14.46

Example 8 A Non-Cook Process for Ethanol Production Using TrGA

In general a 33% slurry of corn flour (Azure Standard Farms) wasprepared in DI H₂O to which 400 ppm urea was added. The pH was adjustedto 4.5. Fermentations were conducted in 125 ml flasks containing 100 gmash and various treatments of GAU/g TrGA. A 20% slurry of Fali dryyeast in water was prepared and mixed with a 32° C. water bath one hourprior to inoculating the fermenters by adding 0.2 ml of the yeastslurry. The flasks were placed in a 32° C. water bath and the mash mixgently. During the fermentations samples were removed for HPLC analysis.The fermentations were terminated after 72 hours. Production ofcompounds including sugars, lactic acid, glycerol and ethanol at varioussampling intervals is shown below in various tables. The mash was driedat 60° C. to obtain the DDGS, and the starch content of the DDGS wasdetermined by the dual enzyme method.

A. All conditions were as described above: the treatment included 1.2GAU/g TrGA.

TABLE 6 Ethanol Production % W/V % W/V % W/V % W/V % W/V % W/V % V/VTreatment Hrs DP > 3 DP-3 DP-2 DP-1 Lactic Glycerol EtOH TrGA 17 0.680.05 0.04 0.00 0.04 0.41 4.70 TrGA 24 0.67 0.06 0.05 0.02 0.04 0.42 5.44TrGA 41 0.65 0.07 0.00 0.00 0.05 0.44 6.78 TrGA 48 0.59 0.08 0.08 0.000.07 0.43 7.77 TrGA 64 0.61 0.08 0.00 0.00 0.15 0.43 8.42 TrGA 72 0.600.08 0.07 0.01 0.17 0.43 8.59

B. All conditions were as described above: the treatments included 0.75GAU/g GA107 and 0.75GAU/g GA104

TABLE 7 Ethanol Production % % % % w/v w/v w/v w/v % w/v % w/v % v/v GAhrs DP > 3 DP-3 DP-2 DP-1 Lactic Glycerol ETOH 1.11 0.10 0.29 1.06 0.000.15 0.00 104 13 0.84 0.00 0.01 0.01 0.00 0.43 5.03 107 13 0.77 0.000.00 0.00 0.00 0.42 4.16 104 21 0.94 0.14 0.03 0.00 0.00 0.46 6.90 10721 0.88 0.10 0.03 0.01 0.00 0.43 5.24 104 35 0.94 0.18 0.13 0.02 0.020.49 9.02 107 35 0.87 0.11 0.02 0.01 0.04 0.44 6.53 104 54 0.91 0.140.00 0.00 0.00 0.51 10.93 107 54 0.89 0.13 0.00 0.00 0.30 0.45 7.58 10462 0.87 0.12 0.00 0.00 0.00 0.53 11.49 107 62 0.88 0.14 0.00 0.00 0.390.46 7.74 104 72 0.94 0.14 0.16 0.00 0.00 0.53 12.22 107 72 0.88 0.140.05 0.01 0.42 0.47 7.82

C. All conditions were as described above: the treatments included a) A.niger GA 0.75 GAU/g+2.25 SSU AkAA and b) TrGA 0.75 GAU/g+2.25 SSU AkAA.The residual starch for AnGA+AkAA treatment was determined to be 5.26%and the residual starch for TrGA+AkAA treatment was determined to be8.71%.

The measurement of alpha amylase activity for AkAA is based on thedegree of hydrolysis of soluble potato starch substrate (4% ds) by analiquot of the enzyme sample at pH 4.5, 50° C. The reducing sugarcontent is measured using the DNS method as described in Miller, G. L.(1959) Anal. Chem. 31:426-428. One unit of the enzyme activity (SSU,soluble starch unit) is equivalent to the reducing power of 1 mg ofglucose released per minute at the specific incubation conditions.

TABLE 8 Ethanol Production % W/V % W/V % W/V % W/V % W/V % W/V % V/VTreatment Hours DP > 3 DP-3 DP-2 DP-1 Lactic Glycerol Ethanol AnGA +AkAA 15 0.81 0.00 0.04 0.13 0.04 0.63 8.22 TrGA + AkAA 15 0.94 0.00 0.040.03 0.04 0.68 8.35 AnGA + AkAA 26.5 0.94 0.06 0.04 0.08 0.06 0.89 12.59TrGA + AkAA 26.5 1.00 0.08 0.08 0.00 0.06 0.83 11.81 AnGA + AkAA 40 0.650.10 0.08 0.05 0.06 0.94 14.37 TrGA + AkAA 40 0.73 0.10 0.14 0.00 0.050.91 13.80 AnGA + AkAA 49 0.93 0.07 0.06 0.05 0.05 1.08 17.05 TrGA +AkAA 49 0.98 0.08 0.14 0.00 0.04 0.97 15.52 AnGA + AkAA 70 0.82 0.040.04 0.27 0.00 1.07 17.59 TrGA + AkAA 70 0.95 0.08 0.04 0.00 0.00 1.0117.17

D. All conditions were as described above: the treatments included a)TrGA 0.695 GAU/g+2.25 SSU AkAA and b) TrGA 0.695 GAU/g+2.25 SSU AKAA+2ASPU/g Pullulanase. One acid stable pullulanase unit (ASPU) is definedas the amount of enzyme which liberates one equivalent reducingpotential as glucose per minute from pullulan at pH 4.5 and atemperature of 60° C.

TABLE 9 Ethanol production % W/V % W/V % W/V % W/V % W/V % W/V % V/VDDGS % Treatment Hours DP > 3 DP-3 DP-2 DP-1 Lactic Glycerol Ethanolstarch TrGA 15 0.92 0.05 0.05 0.04 0.03 0.60 7.69 TrGA + 15 0.91 0.050.04 0.04 0.03 0.60 8.00 Pullulanase TrGA 24 0.94 0.08 0.09 0.05 0.040.72 10.46 TrGA + 24 0.91 0.12 0.10 0.05 0.04 0.73 10.93 PullulanaseTrGA 41 0.91 0.10 0.17 0.05 0.04 0.86 13.89 TrGA + 41 0.92 0.13 0.160.04 0.05 0.87 14.33 Pullulanase TrGA 47 0.87 0.10 0.20 0.05 0.04 0.9014.51 TrGA + 47 0.94 0.13 0.19 0.04 0.03 0.91 15.32 Pullulanase TrGA 700.92 0.11 0.06 0.03 0.00 0.98 17.27 18.5 TrGA + 70 0.95 0.11 0.05 0.020.00 0.98 17.77 16.4 Pullulanase

E. All conditions were as described above: the treatments included TrGA0.695 GAU/g and the following AkAA treatments: a) 3 SSU AkAA; b) 10 SSUAkAA and c) 30 SSU AkAA.

TABLE 10 % Treatment % w/v % w/v % w/v % w/v % w/v % w/v % v/v starch(AkAA) Hours DP > 3 DP-3 DP-2 DP-1 Lactic Glycerol Ethanol DDGS  3 SSU17 0.77 0.05 0.00 0.00 0.04 0.59 8.31 10 SSU 17 0.76 0.04 0.03 0.00 0.030.62 8.85 30 SSU 17 0.78 0.04 0.05 0.00 0.03 0.06 9.54  3 SSU 30 0.740.07 0.05 0.00 0.05 0.73 11.35 10 SSU 30 0.78 0.06 0.05 0.00 0.05 0.8112.62 30 SSU 30 0.85 0.71 0.05 0.03 0.05 0.84 13.91  3 SSU 41 0.70 0.080.02 0.02 0.05 0.90 13.96 10 SSU 41 0.69 0.08 0.02 0.03 0.05 0.91 15.0230 SSU 41 0.68 0.07 0.03 0.07 0.05 0.92 15.83  3 SSU 51 0.73 0.09 0.090.04 0.05 0.98 15.38 10 SSU 51 0.74 0.09 0.05 0.05 0.04 0.99 16.57 30SSU 51 0.73 0.08 0.04 0.03 0.04 0.96 16.53  3 SSU 70 0.70 0.09 0.02 0.020.02 1.04 17.09 15.8 10 SSU 70 0.72 0.08 0.02 0.03 0.04 1.04 17.35 10.730 SSU 70 0.71 0.08 0.02 0.07 0.03 1.01 17.42 9.6

1-21. (canceled)
 22. An isolated enzyme having glucoamylase activity andat least 80% sequence identity with any one of SEQ ID NOS: 17, 18, 19,20, 21, 22, 43, 44, 45, 46 and
 47. 23-27. (canceled)
 28. The isolatedenzyme of claim 22, wherein the enzyme has an amino acid sequence withat least 95% identity to SEQ ID NO:
 17. 29-37. (canceled)
 38. An enzymecomposition comprising a glucoamylase having the amino acid sequence ofclaim
 22. 39-41. (canceled)
 42. The enzyme composition of claim 38,wherein the composition is used in a process to hydrolyze granularstarch.
 43. The enzyme composition of claim 38, wherein the compositionis a detergent composition.
 44. The enzyme composition of claim 38further comprising one or more additional enzymes.
 45. The enzymecomposition of claim 44, wherein the one or more additional enzymes areselected from the group of Aspergillus kawachi alpha amylases,glucoamylases, granular starch hydrolyzing enzymes, proteases,cellulases, pullulanases, phytases and combinations thereof.
 46. Theenzyme composition of claim 45, wherein the additional enzyme is anAspergillus kawachi alpha amylase.
 47. The enzyme composition of claim45, wherein the additional enzyme is a granular starch hydrolyzingenzyme.
 48. The enzyme composition of claim 45, wherein the additionalenzyme is a protease. 49-52. (canceled)
 53. A method of hydrolyzingstarch comprising treating a starch containing substrate with the enzymeof claim 22 or a biologically active fragment thereof.
 54. A method forproducing a fermentation product from a granular starch containingsubstrate comprising. a) contacting a granular starch containingsubstrate with the glucoamylase of claim 22 or a biologically functionalfragment thereof at a temperature below the gelatinization temperature,at a pH of about 4 to 7.0 for a period of time to produce a compositioncomprising glucose, b) contacting the glucose with a fermentationorganism under suitable fermentation conditions to produce afermentation product.
 55. The method of claim 54, wherein thefermentation product is ethanol.
 56. A method for saccharifyingliquefied starch comprising treating liquefied starch with a polypeptidehaving glucoamylase activity, wherein the polypeptide is the enzyme ofclaim 22 or a biologically functional fragment thereof and obtaining acomposition which includes at least 80% glucose.
 57. The enzymecomposition of claim 46, further comprising a protease.
 58. The enzymecomposition of claim 57, further comprising a phytase.
 59. The enzymecomposition of claim 45, wherein the additional enzyme is a phytase.